1. Field of the Invention
The present invention relates to a Lac shuttle vector and uses thereof. More particularly, the Lac shuttle vector features a non-antibiotic resistance gene as a selection marker.
2. Description of the Related Arts
Currently, most DNA vaccines are administrated by the uptake of bacteria such as Salmonella typhimurium or Listeria monocytegenes to mammals (Dietrich G., et al., 1998, Nature Biotech., 16:181–185; Lowrie, D. B., 1998, Nature Med., 4:147–148). The advantage is that the DNA vaccine can be directly incorporated into immune cells, or the immune system can be stimulated by the DNA vaccine, thereby enhancing the immune response. Usually, the effect produced by the administration of such bacteria is better than that produced by using DNA alone as a vaccine. However, such kinds of attenuated bacteria may be harmful to mammals being treated or may become pathogenic due to the mutagenesis of these bacteria.
During genetic engineering, plasmids have to bear a selection marker for selection of a cell (e.g. bacteria) containing the plasmid. Generally, most commercially available plasmids bear the antibiotic resistance gene such as an ampicillin resistance gene or a kanamycin resistance gene as a selection marker. Because most of these plasmids are used in laboratories, safety is not an important concern. However, some bacteria strains present in organisms may contain the antibiotic resistance genes via the natural transduction effect. If these plasmids are used as a pharmaceutical vaccine composition or food additive, organisms treated with the plasmids or the derivatives thereof may be jeopardized. In addition, the use of antibiotics can be problematic because of the potential for residual antibiotics in the purified DNA, which can cause an allergic reaction in a treated organism.
Therefore, the object of the present invention is to construct a vector without antibiotic resistance gene and use a harmless host cell to express heterologous genes in an organism or as a DNA vaccine or health food, thereby raising the safety and enhancing the immune response.
Conventional attenuated bacteria used for DNA vaccines may cause pathogenicity. Therefore, lactic acid bacteria are selected for a vaccine medium in the present invention to achieve the above object. Lactic acid bacteria are Gram-positive bacteria without pathogenicity, and are used in large quantity in the dairy and food industries. In addition, lactic acid bacteria are normal flora present in the digestive tract (Bomba A., et al., 1994, Vet. Med. (praha), 39:701–710; Nemcova R., et al., 1998, DTW Dtsch tierarztl wochenscher, 105:199–200). Further, certain chemical composition in the cell walls of lactic acid bacteria can stimulate the immune response in human (Vilma M. A., et al., 1996, Chem. Pharm. Bull., 44(12):2263–2267). If the lactic acid bacterial strains are modified so as to bear a heterologous gene or to be used as a DNA vaccine, the immunity or health of the organisms can be improved after uptake of such modified lactic acid bacteria. In addition, the recombinant plasmids or vectors can be prepared using genetic engineering to obtain more valuable lactic acid bacterial strains useful in the food industries.